The goal of this proposed study is to develop an effective strategy for induction of durable human organ transplant tolerance using a renal transplant model to serve as a preclinical bridge with direct relevance to clinical organ transplantation. Our preliminary studies in a novel posttransplant strategy (posttransplant rabbit antithymocyte globulin (RATG), subtotal fractionated limited lymphoid irradiation and splenectomy (LLlI/splx), followed by infusion of a subpopulation of donor bone marrow cells (DBMC)) have demonstrated development of specific donor unresponsiveness, chimerism, and acceptance of life-sustaining, MHC class I and II disparate renal allografts (KTx) in rhesus monkeys for >3.5 years with no immunosuppression given before or after the first week posttransplant. Although the results are compelling, preclinical issues persist that require resolution to promote safe application to human transplantation. The proposed studies will examine a hypothesis that early, effective inhibition of direct and indirect pathways of alloantigen presentation by a combined low morbidity therapeutic approach using pluripotential DBM stem cells and short-term recipient immunosuppression will facilitate stable chimerism and tolerance without chronic immunosuppressive therapy. The specific aims of the proposal are: (1) To investigate the capacity of DR-/dim CD3-/dim CD34+ hematopoietic stem cells purified from donor bone marrow (DBM-HSC) to induce stable chimerism and tolerance in short-term immunosuppressed KTx recipients: To accomplish this we will use colony stimulating factors to establish optimal DBM-HSC recovery and culture conditions for ex vivo propagation of DBM-HSC; examine the activity of freshly isolated and precultured DBM-HSC preparations for induction of chimerism and KTx tolerance; determine if stable tolerance induction will require confusion of pure CD34+ DBM-HSC with the CD8+ DBM fraction that mediates veto activity in rhesus monkeys. (2) To examine the role of DBM-HSC derived chimerism in maintenance of tolerance: These studies will (A) examine the lineage and levels of chimeric cells in various tissues in relation to immunological unresponsiveness; (B) directly examine the role of DBM-HSC derived chimerism in maintenance of tolerance using DBM-HSC that are transduced with HSV-1 thymidine kinase before infusion to render them susceptible to subsequent destruction by ganciclovir in the tolerant recipient; (3) To assist transition of this tolerance strategy to clinical organ transplantation: Using the optimal DBM-HSC protocol, we will examine the effects of recipient cytokine pretreatment, timing of DBM-HSC infusion, adjunctive short term immunosuppressive drugs, and mobilized PBLHSC to facilitate clinical application. The information gained in these studies is expected to guide successful application of tolerance induction to human transplantation.